Cardinal symptoms in Parkinson's disease (PD) result primarily from death of dopaminergic (DA) neurons in substantia nigra pars compacta (SNpc) (Dauer et al., Neuron 39, 889-909 (2003); Dawson et al., Science 302, 819-22 (2003)). Current interventions ameliorate symptoms but there is no treatment to halt or delay loss of DA neurons. Several lines of evidence suggest that respiratory chain dysfunction is involved in the pathogenesis of PD (Dauer et al., Neuron 39, 889-909 (2003); Dawson et al., Science 302, 819-22 (2003)): (1) A deficient function of the respiratory chain has been reported in cell lines and tissues from PD patients (Mizuno, Y. et al., Bioch. Biophys. Res. Comm. 163, 1450-1455 (1989); Schapira, Adv Neurol 86, 155-62 (2001)). (2) Certain mitochondrial DNA (mtDNA) polymorphisms reduce the risk of PD (van der Walt, et al., Am J Hum Genet 72, 804-11 (2003)). (3) The toxin MPP+ inhibits complex I of the respiratory chain and it causes parkinsonism after selective uptake in DA neurons (Langston et al., Science 219, 979-80 (1983); Mizuno et al., J Neurochem 48, 1787-93 (1987)). (4) The pesticide rotenone is a strong inhibitor of complex I and long term exposure causes parkinsonism in rats (Betarbet et al., Nat Neurosci 3, 1301-6 (2000)). (5) The herbicide paraquat inhibits complex I and can in combination with other agents cause selective degeneration of DA neurons (Thiruchelvam et al., J Neurosci 20, 9207-14 (2000)). In addition, the mitochondrial respiratory chain is a major producer of ROS and oxidative stress is directly involved in the cascade of biochemical changes causing DA cell death (Thiruchelvam et al., J Neurosci 20, 9207-14 (2000)).
The respiratory chain consists of five enzyme complexes formed by ˜100 different protein subunits that perform the process of oxidative phosphorylation to generate adenosine triphosphate (ATP), which is used as the energy source for a large number of cellular processes (Saraste, Science 283, 1488-93 (1999)). The biogenesis of the respiratory chain is unique in its bipartite dependence on both nuclear and mtDNA genes. The expression and maintenance of mtDNA is completely controlled by nuclear genes, as exemplified by mitochondrial transcription factor A (TFAM) which regulates mtDNA transcription (Fisher et al., J. Biol. Chem. 260, 11330-11338 (1985); Parisi et al., Science 252, 965-969 (1991); Larsson et al., Annu. Rev. Genet. 29, 151-178 (1995); Falkenberg et al. Nat. Genet. 31, 289-294 (2002)) and mtDNA copy number (Ekstrand et al., Hum. Mol. Genet. 13, 935-944 (2004)). We have constructed a conditional knockout allele (Gu et al., Science 265, 103-106 (1994)) by inserting loxP sequences into the Tfam locus to generate TfamloxP animals (Larsson et al., Nat. Genet. 18, 231-6 (1998)). The TfamloxP mice have normal TFAM protein expression and normal oxidative phosphorylation capacity (Wang et al., Nat. Genet. 21, 133-7 (1999)). The protein cre recombinase will specifically recognize and recombine loxP sequences, thereby deleting any DNA between them. By controlling the expression of cre we can in our system choose in what tissue or cell type we want to knock out Tfam. We have extensively documented that this system allows very efficient germ line and tissue-specific knockout of Tfam (Larsson et al., Nat. Genet. 18, 231-6 (1998); Wang et al., Nat. Genet. 21, 133-7 (1999); Li et al., Proc. Natl. Acad. Sci. USA 97, 3467-72 (2000); Hansson et al., Proc Natl Acad Sci USA 101, 3136-3141 (2004); Silva et al., Nat. Genet. 26, 336-340 (2000); Sorensen et al., J. Neurosci. 21, 8082-8090 (2001); Wredenberg et al., Proc Natl Acad Sci USA 99, 15066-71 (2002)). Heterozygous germ line knockout animals (+/Tfam−) have a reduction of mtDNA copy number in all tissues and a moderate respiratory chain deficiency in the heart, whereas homozygous germ line knockouts (Tfam−/Tfam−) die in mid-gestation due to lack of mtDNA and absence of oxidative phosphorylation (Larsson et al., Nat. Genet. 18, 231-6 (1998)). We have also studied pathophysiological events associated with mitochondrial dysfunction by selectively disrupting Tfam in heart muscle cells (Wang et al., Nat. Genet. 21, 133-7 (1999); Li et al., Proc. Natl. Acad Sci. USA 97, 3467-72 (2000); Hansson et al., Proc Natl Acad Sci USA 101, 3136-3141 (2004)), insulin-secreting β-cells (Silva et al., Nat. Genet. 26, 336-340 (2000)), forebrain neurons (Sorensen et al., J. Neurosci. 21, 8082-8090 (2001)) and skeletal muscle cells (Wredenberg et al., Proc Natl Acad Sci U S A 99, 15066-71 (2002)).
A critical gap in our knowledge of PD is a validated and widely acceptable rodent model in which endogenous disruption of respiratory enzymes leads to a PD-like phenotype. An ideal genetic model for PD would have the following characteristics (Beal, Nat Rev Neurosci 2, 325-34 (2001)): (1) Normal number of DA neurons at birth with a gradual loss during adulthood. (2) Easily detectable motor deficits including bradykinesia, rigidity and resting tremor. (3) Histology showing typical α-synuclein pathology including formation of the intracellular inclusions known as Lewy bodies. (4) Robust genetics allowing easy propagation of the genotype. (5) A progressive disease course of just a few months for rapid testing of different therapeutic strategies.
Almost all available in vivo models of PD are based on the administration of neurotoxins to animals. The first agent used to produce an animal model of PD was 6-OHDA (Ungerstedt, Acta Physiol Scand Suppl 367, 69-93 (1971)). This toxin is normally injected unilaterally directly into the substantia nigra, the medial forebrain bundle or the striatum causing a selective loss of DA neurons. 6-OHDA lesions do not result in typical Lewy body formation and can cause nonspecific damage to other neurons. When injected into the substantia nigra 6-OHDA treatment is acute, causing a rapid cell loss not resembling the progressive nature of PD. If injected into striatum the effect is slower (<4 weeks) but then only partial. This limits researchers to study only the end-stage or a very short progression of the disease (Beal et al., Nat Rev Neurosci 2, 325-34 (2001); Ungerstedt, Acta Physiol Scand Suppl 367, 69-93 (1971); Deumens et al., Exp Neurol 175, 303-17 (2002)).
The most widely used animal model of PD today is acute systemic administration of MPTP which in primates causes parkinsonism with a selective loss of DA neurons in the substantia nigra. Rodents are much less sensitive to MPTP and require a higher dose while the typical behavioral symptoms of parkinsonism rarely appear. The main disadvantages of MPTP is the resistance of most rodents and the acute nature of the model preventing researchers from studying the progression of the disease (Shimohama et al., Trends Mol Med 9, 360-5 (2003)).
Recently it was discovered that systemic treatment with low doses of the naturally occurring pesticide rotenone over time causes parkinsonism in rats. Treated animals show a selective loss of DA neurons in the substantia nigra associated with intracellular inclusions similar to Lewy bodies. The outcome of rotenone treatment is highly variable as only a part of the treated rats develop PD-like pathology and some strains do not respond at all to the drug (Betarbet et al., Nat Neurosci 3, 1301-6 (2000)). All these pharmacological models also suffer from the inherent risk of other pleiotropic effects of systemic drug administration.
In the present document we describe the generation of a mouse with a specific disruption of Tfam and hence oxidative phosphorylation in DA neurons only. We use the dopamine transporter (DAT) locus to direct the expression of cre recombinase to this specific cell type. Such mice over the time of several months develop a PD-like behavioral phenotype associated with loss of DA neurons in the SNpc and pathology typical for the disease. The generated animal model faithfully reproduces key pathophysiological features of PD, i.e. slow progressive loss of DA terminals in striatum and loss of DA neurons in SNpc; α-synuclein reactivity including intracellular inclusions similar to Lewy bodies in affected areas prior to and during cell loss; progressive movement disorder, that is partially reversed by L-DOPA treatment, associated with abnormal gait, tremor and rigid limbs. This model will be a valuable tool for testing pharmacological, gene and cell therapies to counteract or cure PD.
As used herein, the term “genetically modified” refers to any purposeful alteration of the naturally-occurring genome of an animal. For example, sequences can be inserted into the genome which, when activated, can cause selective deactivation of a particular gene adjacent to the inserted sequence.
The term “dysfunction” refers to any deviation from normal or naturally-occurring function in a disease-free state.
The term “naturally exhibiting” refers to the characteristics or traits occurring under normal environmental and physiological conditions.
As used herein, the term “Tfam” relates to mitochondrial transcription factor A (Fisher et al., J. Biol. Chem. 260, 11330-11338 (1985); Parisi et al., Science 252, 965-969 (1991); Larsson et al., Annu. Rev. Genet. 29, 151-178 (1995); Falkenberg et al. Nat. Genet. 31, 289-294 (2002)) and mtDNA copy number (Ekstrand et al., Hum. Mol. Genet. 13, 935-944 (2004)).
As disclosed herein, the terms “Parkinson mouse” and “homozygous knockout animals” both relate to transgenic mice comprising the 5′ end of the dopamine transporter gene fused to a DNA sequence encoding cre recombinase as well as a DNA sequence comprising a sequence encoding Tfam which sequence also contains LoxP sequences (+/DAT-cre; TfamloxP/TfamloxP). This recombination leads to cell type specific homozygous disruption of Tfam and hence oxidative phosphorylation in DA neurons only, and accordingly such mice display symptoms of Parkinson's disease.